Cholera has been known for many centuries as a deadly disease whose victims die after bouts of severe diarrhea from dehydration and malnutrition. The causative organism replicates in the intestine. In response to the toxins produced, the intestine excretes large amounts of water. However, in tissue culture, cholera toxin stimulates growth of epithelial cells. (Taylor-Papadimitriou, et al., J. Cell. Physiol. 102: 317-321 (1980) and Chopra, et al., Gastroenterology 92: 891-904 (1987)) It had, furthermore, been known that growth of some malignant cells was inhibited by treatment with cholera toxin. (Cho-Chung, et al. Cancer Res. 43: 1473-1476 (1987)). It was assumed, by Cho-Chung and her co-workers that the response to the toxin was due to stimulation of the intracellular cAMP system. (Her work was primarily concerned with mammary cancers and lymphomas.) However, the work of Cho-Chung did not lead to a means of treating malignancies in patients, since no means of predicting efficacy in treating a particular patient could be predicted and no method for therapeutic treatment was described.
The treatment of human small cell lung carcinoma (SCLC), has heretofore been difficult. The disease accounts for about 30% of all lung cancers. The malignancy is characterized by several neuroendocrine features which include the elaboration of peptide hormones such as gastrin-releasing peptide (GRP). The growth of the cells in serum free conditions is often enhanced in the presence of bobesin.
Previous studies in murine fibroblasts, SCLC, anterior pituitary, and rat insulinoma cells have shown that binding of GRP to its receptor results in activation of phospholipase-C, increased phosphatidylinositol (PI) turnover, release of calcium from internal stores, increase of intracellular free calcium ([Ca2+]i), and activation of protein kinase-C. Thus, an understanding of bombesin or GRP stimulation of SCLC growth requires the definition of elements important in the process of bombesin-mediated signal transduction.
As previous studies in multiple tissues have suggested that hormonal activation of phospholipase C may be mediated by a guanine nucleotide binding protein (G protein), a study was begun to evaluate the effects of bacterial toxins known to modulate G protein function on GRP stimulated signal transduction pathways in the SCLC cell line NCI-H345. It was found that pretreatment with cholera toxin (CT) at 1 ug/ml for 18 hours resulted in a substantial inhibition in basal and GRP stimulated intracellular calcium and inositol trisphosphate (IP.sub.3), as well as the production of GRP stimulated inositol monophospates.
The classical biology of CT did not readily account for the observations of Cho-Chung and others. CT binds to a cell surface glycolipid, the G.sub.M1 ganglioside, through its beta subunit. After internalization, the pentameric beta subunits are cleaved from the catalytic alpha subunit which in turn stimulates the ADP ribosylation of Gas, the stimulatory G protein coupled to adenylate cyclase, leading to an inhibition of its GTPase activity. This non reversible GTP-bound state in turn activates adenylate cyclase and leads to an elevation of intracellular cAMP levels. However, reproduction of the effects of CT on GRP mediated signal transduction using other maneuvers to increase intracellular cAMP was not possible.